Although much attention has been devoted to understanding the pathogenic attributes that distinguish one bacterial species from another, less is known about the causes of intra-species differences in virulence. The question of why some strains of a particular bacterial species cause more severe infections than others has important clinical implications in that it would allow physicians to identify and target patients infected wih particularly aggressive strains and at risk for especially poor outcomes. In this application, we focus on the bacterial pathogen Pseudomonas aeruginosa (PA) to address this question. As with most bacteria, PA has a genome that can be divided into two parts: the core genome, which consists of those genes present in all PA bacteria, and the accessory genome, which consists of those genes found in some strains of PA but not others. Although a substantial amount of work has been performed on characterizing virulence determinants encoded by the PA core genome, the accessory genome remains relatively unexplored. We hypothesize that the accessory genome of PA contains novel virulence genes that impact the severity and type of infection caused by this bacterium. In preliminary work, we have used a comparative genomics approach to identify virulence genes in the PA accessory genome. In this application, we propose to characterize the mechanisms of several virulence factors uncovered by this approach. Our goal here is to demonstrate the accessory genome's potential as a reservoir for virulence factors with interesting mechanisms of action. We will also use our PA database and our comparative genomics approach to identify accessory genes that allow PA to cause specific disease manifestations. To accomplish this, we will use subsets of PA isolates associated with two distinct types of infection: Shanghai Fever and cystic fibrosis. Finally, we will show the versatility of this comparative genomics approach by using it to identify accessory genes that explain why some PA strains are more cytotoxic than others. Completion of these aims will further define the importance of the accessory genome in PA pathogenesis and identify novel virulence determinants that confer different infection phenotypes on strains within the PA species. This work will also lay the foundation for future studies aimed at determining whether these genes constitute genomic fingerprints for particularly aggressive PA strains.